Melatonin and its protective role in attenuating warm or cold hepatic ischaemia/reperfusion injury

Abstract Although the liver is the only organ with regenerative capacity, various injury factors induce irreversible liver dysfunction and end‐stage liver disease. Liver resection and liver transplantation (LT) are effective treatments for individuals with liver failure, liver cirrhosis and liver cancers. The remnant or transplanted liver tissues will undergo hepatic ischaemia/reperfusion (IR), which leads to oxidative stress, inflammation, immune injury and liver damage. Moreover, systemic ischaemia induced by trauma, stroke, myocardial ischaemia, haemorrhagic shock and other injury factors also induces liver ischaemia/reperfusion injury (IRI) in individuals. Hepatic IRI can be divided into warm IRI, which is induced by liver surgery and systemic ischaemia, and cold IRI, which is induced by LT. Multiple studies have shown that melatonin (MT) acts as an endogenous free radical scavenger with antioxidant capacity and is also able to attenuate hepatic IRI via its anti‐inflammatory and antiapoptotic capacities. In this review, we discuss the potential mechanisms and current strategies of MT administration in liver surgery for protecting against warm or cold hepatic IRI. We highlight strategies to improve the efficacy and safety of MT for attenuating hepatic IRI in different conditions. After the potential mechanisms underlying the interactions between MT and other important cellular processes during hepatic IR are clarified, more opportunities will be available to use MT to treat liver diseases in the future.

trauma, stroke, myocardial ischaemia, haemorrhagic shock and other injury factors. 2 Pre-existing liver diseases not only directly lead to a high frequency of liver surgery but are also positively correlated with the severity of hepatic IRI. 3 Moreover, obesity, older age and alcoholism can easily impair liver regenerative capacity and result in hepatic steatosis, which is associated with a higher complication index and postoperative mortality after liver surgery. 4 Hepatic IRI can be divided into warm IRI, which is induced by liver surgery and systemic ischaemia, and cold IRI, which is induced by LT. 5 Cessation of hepatic blood supply is always carried out by clamping manoeuvres and inevitably exposes the liver to IR and leads to liver dysfunction in mammals, while restoration of blood flow further exacerbates liver damage to ischaemic liver tissues. 6 In LT, excised liver grafts are stored in cold preservation solutions before LT, which leads to cold IRI in transplants. 7 Melatonin (MT), namely, N-acetyl-5-methoxytryptamine, was first found to be synthesized from the amino acid tryptophan in the pineal gland and participates in the regulation of sleep promotion, circadian rhythms and neuroendocrine processes. 8 In addition, MT participates in the regulation of energy metabolism, immune function, cardiovascular function, sexual behaviour, the neuropsychiatric system and reproduction. 9,10 It serves as a potent endogenous free radical scavenger that protects against mitochondrial damage and has beneficial effects on tissue injury by clearing reactive oxygen species (ROS) or reactive nitrogen species (RNS) in vitro and in vivo. 11 In liver tissue, MT was reported to protect against oxidative damage and IRI via upregulation of glutathione (GSH) levels, maintenance of mitochondrial membrane structure and reduction of lipid peroxidation, oxidized glutathione (GSSG) levels and polymorphonuclear infiltration. 12,13 MT and its metabolites resist inflammation and prevent disturbances in mitochondrial redox reactions, biogenesis, dynamics and mitophagy to further protect against hepatic IRI. [14][15][16] In this review, we discuss the potential mechanisms and current strategies of MT administration in liver surgery for protecting against warm or cold hepatic IRI. Although MT is considered an effective agent with protective effects against hepatic IR, we still highlight strategies to improve the efficacy and safety of MT for attenuating hepatic IRI in different conditions. After the potential mechanisms underlying the interaction between MT and other important cellular processes during hepatic IR are clarified, more opportunities will be available to use MT to treat clinical liver diseases in the future.

| MITOCHONDRIAL DYS FUN C TION , INFL AMMATI ON AND IMMUNE RE S P ON S E S IN HEPATI C IR
Hepatic IR results in liver injury via activation of mitochondrial ROS, inflammation and immune responses ( Figure 1). Depletion of blood flow in liver tissue transforms hepatocyte metabolism into anaerobic respiration and disrupts oxidative phosphorylation. Ischaemia limits the mitochondrial electron transport chain and further leads to deposition of electron carriers at the onset of reperfusion. 17 A lack of oxygen supply results in parenchymal cell death as a consequence of metabolic disturbances, including glycogen consumption, adenosine triphosphate (ATP) depletion, xanthine oxidase conversion and intracellular pH reduction. 18 Liver ischaemia also results in imbalances in Ca 2+ , H+ and Na+ homeostasis and mitochondrial depolarization and finally swelling of sinusoidal endothelial cells (SECs) and Kupffer cells (KCs). 19 Reperfusion reintroduces oxygen into ischaemic tissue, and reperfusion injury is initiated by direct and indirect cytotoxic mechanisms such as oxidative stress injury, inflammation and immune cell recruitment. 20 Oxidative stress promotes the generation of ATP metabolites accompanied by upregulation of superoxide radicals, hydrogen peroxide and hydroxyl radicals. 21 The adaptive immune response and early and massive T-cell recruitment were initiated after the preservation of ischaemic liver grafts in cold stock solution. 22 Following reperfusion in LT recipients, liver grafts easily develop primary nonfunction or impaired primary function in which they undergo microcirculatory dysfunction and pH homeostasis disturbance. [23][24][25] IR induces oxidative stress, which causes mitochondrial permeability transition pore (MPTP) opening, resulting in loss of mitochondrial membrane potential, mitochondrial swelling and decreased ATP generation. 26 Oxidative stress further leads to the generation of ROS, inflammatory cytokines, and complement factors and the upregulation of autophagy, endoplasmic reticulum (ER) stress and mitochondrial dysfunction. 5 Although mitochondrial or cytosolic ROS are unable to directly cause cytotoxicity in liver cells, they promote lipid peroxidation and stimulate the release of damage-associated molecular patterns (DAMPs), namely, nuclear protein high-mobility group box 1 (HMGB1). In response to Tolllike receptor 4 (TLR4) on the surface of KCs, HMGB1 migrates from hepatocytes and binds to KCs to activate sterile inflammation and promote the generation of additional ROS. 27 Lipid peroxidation further upregulates the release of cytochrome c into the cytoplasm, the activation of caspases and the initiation of cell death after the upregulation of mitochondrial membrane permeability and the loss of mitochondrial integrity. 28 Reperfusion-induced inflammation has been reported to induce hepatic IRI in parenchymal and nonparenchymal cells from in situ and transplanted liver grafts. 20,29 Various inflammatory cells, such as neutrophils, KCs, T lymphocytes, natural killer T (NKT) cells, and various humoral factors, such as complement factors, cytokines and chemokines, are activated to exacerbate injury in SECs and hepatocytes in hepatic IR models. 30 KCs, which are the resident antigenpresenting macrophages in liver tissue, can be first activated for the regulation of downstream inflammatory cells in circulation at the earliest stages of IR. 31 KCs recognize circulating DAMPs to translate alarm signals into an overt inflammatory response. At the initial phase of reperfusion, KC activation enhances the generation of ROS 32 and promotes the release of inflammatory cytokines such as tumour necrosis factor alpha (TNFα), interleukin (IL)-1 and IL-6. After that, IL-1 and TNFα activate CD4 + T lymphocytes to generate TNFβ, TNFγ and granulocyte-macrophage colony-stimulating factor (GM-CSF). 33 In contrast, the pathophysiology of hepatic IR is related to the level of nitric oxide (NO), which is produced by two synthase isoforms, endothelial NO synthase (eNOS) and inducible NO synthase (iNOS). Hepatic SECs constitutively and exclusively produce small amounts of NO to maintain endothelial function for short intervals, and eNOS contributes to the protective mechanism of the endothelium. However, inflammatory factors significantly increase the activity of iNOS to synthesize large amounts of NO and produce free radicals in hepatocytes, SECs, KCs and hepatic stellate cells (HSCs) for sustained periods. 34 Circulating inflammatory factors also trigger the migration of nonresident neutrophils and monocytes into injured sites and initiate a second wave of ROS/RNS production that exacerbates hypoperfusion and the release of proteases, leading to hepatocyte toxicity. 35,36 At the late phase of perfusion, recruitment of neutrophils promotes the release of other inflammatory factors from injured sites, 32 which leads to granulocyte accumulation in the sinusoidal space and microcirculatory disturbances. 37

F I G U R E 1 Hepatic ischaemia/reperfusion results in liver injury via activation of mitochondrial reactive oxygen species (ROS), inflammation and immune responses
Increased damage or intracellular ATP depletion converts the cell death mechanism from apoptosis to necrosis. 40,41 Mitochondriaderived ROS increase lipid peroxidation and mitochondrial membrane permeability, ultimately leading to the release of cytochrome c and caspases and the exacerbation of apoptotic cell death. 28 NO production and inflammatory cytokines also disrupt liver microcirculation and mitochondrial function and promote the generation of caspases, cytochrome c and antiapoptotic protein B-cell lymphoma 2 (Bcl-2) to promote hepatocyte necrosis and liver regeneration. [42][43][44] In particular, three types of autophagy, macroautophagy, microautophagy and chaperone-mediated autophagy (CMA), serve as self-digestion methods to remove long-lived proteins, damaged organelles and malformed proteins to supply ATP and nutrients in mammals. 45,46 Autophagy is reported to be regulated by different signalling pathways, such as the mammalian target of rapamycin (mTOR), mitogen-activated protein kinase (MAPK), Bcl-2 and p53 pathways. 47 The mTOR signalling pathway is the main potent proinflammatory regulator that negatively regulates autophagy activation. 48 Although autophagy can protect against hepatic IRI by counterbalancing ATP deprivation at the ischaemia stage, there is also evidence indicating that sustained and excessive activation of autophagy leads to the progression of cell death at the reperfusion phase. 49,50 The contradictory effects may be attributed to the fact that different types of IR and different liver conditions lead to different degrees of autophagy activation. To compensate for the lost liver function, all these pathophysiological changes convert injured livers to a regenerative state. Stathmin-mediated mitosis was reported to be activated to promote hepatocyte proliferation around the perivascular regions after IR, 51 and several nonparenchymal cells, such as KCs and HSCs, worked together to participate in liver tissue remodelling. 52,53 When regeneration is unable to counteract liver injury induced by excessive injury factors, irreversible liver damage will develop into acute or chronic liver failure. As the potential mechanisms underlying hepatic IRI are complex, targeting individual mechanisms makes it difficult to achieve the desired protection in attenuating hepatic IR-induced liver injury.

| THE ME TABOLIC AND S I G NALLING PATHWAYS OF MT IN LIVER TISSUE
The concentration of MT in liver tissue may depend on hepatic metabolic requirements, and this hormone is probably synthesized in the liver and intestinal tract. 54,55 The level of MT is generally highest in serum, while Lahiri et al documented that the second highest level of MT was in the liver. 56 The gastrointestinal tract releases MT into the portal circulation and liver tissue; moreover, new data suggest that another source of MT is synthesis by the liver. 55 MT is metabolized by cytochrome P450 in liver tissue and is transformed into 6-hydroxymelatonin and N(1)-acetyl-N(2)-formyl-5-methoxykynuramine (AFMK). MT and its metabolites protect against hepatic IRI by directly or indirectly inhibiting oxidative stress, inflammation and immune responses ( Figure 2). However, severe oxidative stress promotes the nonenzymatic MT metabolism via its interaction with ROS and NOS. 57 In the subcellular milieu of liver tissue, the MT concentration is highest in the cell membrane since the cell membrane acts as a reservoir of MT.
Whenever MT is needed in other subcellular circumstances, MT is transferred from the cell membrane into the cell and from the cytosol to the mitochondria and nucleus. The transport mechanism in the subcellular milieu enables MT to have low toxicity when it is administered in high doses. 58 Reiter and colleagues found that the concentration of MT in mitochondria isolated from rat hepatocytes greatly exceeded that in blood; moreover, pinealectomy did not decrease the level of MT in hepatocytes. This enables MT to act as a paracrine or autocrine factor to regulate intracellular events. 59 Melatonin also protects against oxidative stress in liver IRI through multiple novel signalling pathways, such as the TLR, haem oxygenase-1 (HO-1) and c-Jun N-terminal kinase (JNK) pathways. 38 Among the numerous pathways, crosstalk between MT and the TLR system in hepatic IR is pivotal. TLRs are pattern-recognition receptors that recognize conserved pathogen-associated molecular patterns (PAMPs). TLR4 is one of the putative HO-1 repressors in noninfectious hepatic IR, which indicates that the crosstalk between HO-1 and the TLR4 system is also important in hepatic IR. 60

| MT TRE ATMENTS PROTEC T AG AIN S T HEPATI C IRI
Characterization of hepatic IR may unlock a novel therapeutic avenue in which ROS overexpression, and sterile inflammatory disorder can be targeted to preserve liver function during the progression of liver IR. MT was recently recognized as an effective factor in preserving liver function in liver grafts undergoing hepatic IR via its antioxidation, anti-inflammation and antiapoptotic capacities (Table 1).

| MT TRE ATMENT OF WARM IR
Melatonin was reported to protect against IRI by improving mitochondrial respiration, ATP synthesis, mitochondrial swelling and lipid peroxidation in liver tissue. 26    conditions. After the potential mechanisms underlying the interaction between MT and other important cellular processes during hepatic IR are clarified, more opportunities will be available to use MT to treat liver diseases in the future.

ACK N OWLED G M ENTS
This work was supported by the National Natural Science Foundation

CO N FLI C T S O F I NTE R E S T
The authors declare that they have no conflicts of interest.

AUTH O R CO NTR I B UTI O N S
Lanjuan Li contributed to the conception of this study. Chenxia Hu and Lingfei Zhao drafted the manuscript. Chenxia Hu and Fen Zhang revised the manuscript. All authors read and approved the final manuscript.

PE E R R E V I E W
The peer review history for this article is available at https://publo ns.com/publo n/10.1111/cpr.13021.

DATA AVA I L A B I L I T Y S TAT E M E N T
Data sharing is not applicable to this article as no new data were created or analysed in this study.